The diffuse endocrine cells of the gut epithelium comprise the largest endocrine organ in the body and serve vital physiologic functions such as gastric emptying and motility, pancreatic and biliary secretion, and growth and repair of gut epithelium. Gastrin, a peptide hormone secreted by G cells of the antral stomach, induces acid secretion and is a key determinant of stomach pH. Little is known about gut endocrine differentiation, particularly in the stomach, although transcriptional and signaling pathways are beginning to come in focus. The homeodomain protein Nkx6.3 controls terminal differentiation of G cells as well as somatostatin-producing D cells in the stomach antrum;gastrin and somatostatin levels are respectively elevated and reduced in Nkx6.3-null mice. Although the close homolog Nkx6.2 overlaps in expression with Nkx6.3, a function for Nkx6.2 has not been established in the stomach. The aims of this proposal are to characterize the individual and joint functions of Nxk6.2 and Nkx6.3 in gastric endocrine cell differentiation and to define the lineage pathway through which Nkx6.3 specifies G cells. A central hypothesis is that G and D cells differentiate from a common precursor and that alternative cell fates in this endocrine lineage are determined by the amount of Nkx6.3, with high levels promoting G-cell differentiation. I will also examine if Nkx6.2 has similar endocrine cell-specifying properties and might compensate for Nkx6.3 when necessary. Accomplishment of these aims will require a breadth of techniques, including RNA in situ hybridization, immunohistochemistry, quantitative RT-PCR, and creation and analysis of a targeted mouse line expressing tamoxifen-inducible Cre recombinase from the endogenous Nkx6.3 locus. Thus, the proposed studies will not only shed light on molecular mechanisms of gastric endocrine cell specification but also expose me to a range of new experimental approaches and laboratory methods. Taken together, these studies will help define the differentiation pathways for gastric epithelial endocrine cells and extend my training for a research career in academic pediatric gastroenterology. PUBLIC HEALTH RELEVANCE: Cell lineage specification and differentiation occur through complex transcription factor cascades, and some gut transcription factors are linked to cancer and other disease;likewise, dysregulation of gut endocrine cells may lead to disorders of acid secretion, motility or digestion. Nkx6.3 seems to act in the final specification steps for G cells, which signal acid production in the stomach. This work will trace the pathway through which Nkx6.3 specifies G cells and also determine if the sibling factor Nkx6.2 has similar functions and/or compensate for absence of Nkx6.3.